Detection of Somatic TP53 Mutations in Tampons of Patients With High-Grade Serous Ovarian Cancer

Introduction

Unlike other gynecologic malignancies, epithelial ovarian cancer typically presents at an advanced stage. This is in part due to the fact that no effective screening methods exist to detect early stage disease and patients with advanced stage ovarian cancer usually have nonspecific symptoms at the time of diagnosis. Thus, despite modest improvements in treatment of advanced ovarian cancer, most patients eventually succumb to their disease.

To date, no effective serum biomarker or imaging based strategy has proven to reduce mortality related to ovarian cancer. As an alternative to these screening approaches, identifying tumor cells through detection of somatic mutations may provide a different method of early cancer detection. The vast majority of epithelial ovarian tumors of the serous histologic subtype harbor TP53 mutations (1, 2). Given that the intra-abdominal cavity communicates with the vagina through the upper genital tract, we speculated that we could detect ovarian cancer cells that exfoliate and descend through the cervical os and into the vagina. We considered the possibility that malignant cells that have exfoliated from the tumor might be detected by deep sequencing of TP53 exoms, which would allow for the detection of even a small fraction of mutant DNA (as little as 0.001%) within the context of a majority of wild-type alleles present in the DNA sample (3). In this study, we hypothesized that if these tumor cells and fragments containing tumor DNA are present in the vagina of women with known ovarian cancer, they could be collected using a tampon.

Materials and Methods

The study was approved by the Institutional Human Subjects Protection Review Board at the University of Alabama at Birmingham and Johns Hopkins Hospital and carried out in accordance with their standards. Patients were approached for enrollment at the gynecologic oncology clinic from August 2012 through January of 2013. Eligible participants included patients with a pelvic mass suspicious for malignancy and planned diagnostic or therapeutic surgery. Exclusion criteria included previous hysterectomy or bilateral salpingo-oophorectomy, age younger than 19, heavy vaginal bleeding, inability or unwillingness to place a vaginal tampon. Patients with serous carcinoma of the ovary comprised the study group for this report.

After obtaining informed consent, patients were given a commercially available plastic applicator vaginal tampon (Tampax Pearl). Patients were instructed to place the tampon in their vagina 8–12 hours prior to their scheduled surgery. The tampon was removed in the operating room following induction of anesthesia and placed in a sterile phosphate buffered saline (PBS) buffer solution. Tumor specimens were collected at the time of surgery from either the primary or metastatic site and immediately snap frozen in liquid nitrogen and stored at −80°C. Because we hypothesized that patients with tubal intraepithelial carcinomas may be more likely to have detectable malignant cells in the vagina, all fallopian tubes were subjected to thin sectioning with the SEE-FIM protocol {Mingels, 2013 #5142}.

The tampons were manually compressed into sterile PBS solution and then discarded. The remaining suspension was then centrifuged at 8000 RPM for 2 minutes and DNA extracted from the cell pellet using a modified protocol and reagents from the QIAamp DNA mini kit (Qiagen). The cell pellet was resuspended in 500 µl of Buffer AE (Qiagen) then pelleted for 1 minute at 8,000 rpm (6941g). The supernatant was discarded and the pellet suspended in 300 µl of Buffer ATL and 30 µl of Proteinase K, pulse vortexed for 10 seconds, then incubated at 56°C for 60 minutes with a 10 second vortex every 15 minutes. Post incubation, 300 µl of Buffer AL plus 560 µl of molecular grade 100% ethanol was added and mixed by vortex. The solution was then transferred to a QIAamp Mini spin column, spun at 8000 rpm for 75 seconds and the follow-through discarded. The column was washed with 500 µl of Buffer AW1, centrifuged at 8,000 rpm (6941g) for 2 minutes, then washed with 500 µl of Buffer AW2 and centrifuged at 10,000 RPM (10,845g) for 2 minutes. The column was dried by centrifugation at 15,000 RPM (24,400g) for 3 minutes. DNA was eluted using 50 µl of Buffer AE by centrifugation after 5-minute room temperature incubation. DNA concentration and 260/280 ratios were quantified using a micro volume spectrophotometer (Biotek Epoch).

For tumor specimens, a small portion of snap frozen tumor immediately adjacent to H&E-confirmed malignant tissue was thawed and 10–20 mg of tumor was processed for DNA extraction using the DNEasy kit (Qiagen) as instructed by the manufacturer.

A sequencing error-reduction technology described previously {Kinde, 2013 #5141} was used to sequence TP53 in DNA from the tumor and tampon samples. Amplification primers were designed to amplify segments containing all exons of the TP53 gene. The PCR products were purified with AMPure and sequenced on a MiSeq instrument. Data were collected and analyzed as previously described (3).

Descriptive statistics were used to describe mutation rates and frequencies. Fisher’s exact test was used to measure association between variables. A p value of <0.05 was considered significant.

Results

During the study period, 33 patients were enrolled into the trial. Tampons were successfully placed and processed in 25 patients (76%, 95% CI 58%–89%). Reasons for failed collection included patients forgetting or electing not to place the tampon, the tampon falling out prematurely, and incorrect application. Adequate quantity and quality of DNA was obtained from all patients who successfully placed the tampon and had it removed preoperatively. Of the 25 patients, 13 had benign disease, 3 had non-ovarian malignancies (uterine carcinosarcoma and appendiceal carcinoma), and 9 patients had serous (ovarian, tubal or primary peritoneal) adenocarcinoma. One patient with serous adenocarcinoma had inadequate DNA obtained from her primary tumor. Thus, for this initial analysis, DNA from the tumor specimens from the eight patients with serous carcinoma and adequate DNA samples were analyzed for the presence of TP53 mutations in any coding exon. The mutation identified in the tumor was then queried in the corresponding tampon DNA sample using the same technique. The clinical characteristics of the 8 patients examined are listed in Table 1. One patient had identifiable tubal intraepithelial neoplasia. Of note, 3 patients had bilateral tubal ligations.

Information regarding TP53 mutational analysis is demonstrated in Table 2. The average amount of DNA recovered from the vaginal tampon was 133.2ng/µl (range 14 to 609 ng/µl; Table 2). No clinical factors correlated with the amount of DNA recovered from the tampons, including length of time the tampon was in the vagina. Tumor specimens from all 8 patients showed at least one mutation in TP53.

The sequencing method not only allows identification of mutations, but also quantification of the number of DNA fragments containing the mutation. Interestingly, although all tumor specimens contained at least 50% of malignant cells in the tumor, generally mutated DNA in the specimens made up a smaller percentage of the total DNA, suggesting that histologic assessment may overestimate the overall makeup of malignant cells in a heterogeneous tumor.

Mutational analysis of the tampon specimen DNA revealed mutations in 3 of the 8 patients (38%, 95% CI 9%–76%). No mutations were observed in the tampon DNA of the three patients who had undergone tubal ligation, while mutations in the tampon DNA were observed 3 of the 5 patients (sensitivity 60%) without tubal ligation (0% vs 60%, p=0.20). The mutation identified in the tampon DNA was the same mutation identified in the tumor in all three patients (Table 2). The fraction of mutant alleles in the tampon DNA, which approximates the percentage of vaginal DNA that was tumor derived, ranged from 0.01% to 0.07%. The fraction of mutant alleles in the corresponding tumors varied from 32% to 59%.

There was insufficient statistical power to evaluate the relationship between any clinical parameters (including age, race, tumor characteristics, CA-125, presence of ascites, length of time of tampon placement) and ability to detect of TP53 mutations (data not shown).

Discussion

In this pilot study, vaginal tampons were used to collect DNA from women newly diagnosed with advanced papillary serous ovarian cancer. Deep sequencing of this vaginal DNA yielded detectable TP53 mutations in 60% of serous carcinoma patients without a history of tubal ligation. TP53 mutations were not detectable in the vaginal DNA obtained from all 3 serous carcinoma patients with a prior tubal ligation. The TP53 mutations detected in the tampon DNA samples were identical to the mutations found in the primary metastatic tumor. This supports the hypothesis that ovarian cancer cells survive transit through the fallopian tube, uterus and reach the cervix and vagina intact (8). We postulate that the exfoliated cells from the primary ovarian malignancy (ovarian, tubal or primary peritoneal) migrate through the fallopian tubes, the uterus and then through the endocervix and into the vagina.

Recent research suggests that a majority of serous ovarian cancers appear to originate from dysplastic lesions in the distal fallopian tube (4, 5). Vaginal tumor DNA was not detected in the one patient with tubal intraepithelial neoplasia (TIN). The rate of identifiable TIN was lower in this cohort than in previously published studies, however the diagnosis of TIN is still variable among pathologists in different institutions. With only one patient with intraepithelial neoplasia, we cannot make any specific conclusions regarding the feasibility of this method in patients with early stage or pre-invasive disease.

This study gives further support to the potential utility of ultrasensitive genomic analysis for the detection of low frequency mutations in clinical samples. In a recent study utilizing the same sequencing technology, Pap smear solvents (rather than tampons) in 22 patients with ovarian cancer were analyzed (8). Nine of these 22 patients had detectable TP53 mutations in the DNA from the Pap smear fluids (9/22, 41%). It is possible that the sensitivity of this approach might be improved by the use of a method that potentially collects both descending tumor cells and tumor derived fragments (e.g. exosomes) reaching the vaginal cavity.

The fraction of mutant alleles in the liquid based Pap smear solvent was higher (median 3%, range 0.01 to 80%) than that observed in the current study with tampon extracts (range 0.01 to 0.07%). The advantage of tampons, however, is that they do not require a medical professional for sample procurement and can be more easily used for serial sampling. Frequent, serial sampling, especially in selected high risk populations, can significantly improve the predictive value of diagnostic tests such as Cancer Antigen 125 (CA-125) in women with germline BRCA mutations. Our approach takes advantage of relatively non-invasive sampling and uses materials that would routinely be collected and discarded, but could potentially be shipped to a central laboratory for testing. For this method to ultimately be clinically useful, several factors should be considered. The cost of the sequencing analysis of TP53 is currently not amenable to a screening setting; however, it is anticipated that costs of next generation sequencing methods will continue to drop exponentially. Most importantly, this approach will have to be shown to be able to adequately detect early stages of disease to provide sufficient lead-time for an effective intervention.

One limitation of the current study was that all samples were obtained from patients with late-stage cancer. Because many serous cancers may originate in the fallopian tube in precancerous tubal intraepithelial carcinomas, it may be feasible to detect precancerous disease or cancer prior to metastatic spread outside of the genital tract with this next generation sequencing method (4). Detection of preinvasive disease has been modeled in other tumor types, most notably the detection of preinvasive colorectal cancer through fecal DNA testing (6). Another limitation is that we did not sequence the DNA from tampons from patients with benign disease. Therefore, specificity could not be calculated.

In summary, the current study suggests that tumor DNA reaches the vagina in advanced ovarian cancer patients and can feasibly be collected using tampons and detected therein using high throughput TP53 mutational analysis. The majority of patients with ovarian cancer present at an advanced stage because screening and early-stage diagnostic methods are imprecise, and none can currently be recommended for screening. This method may represent an innovative way to detect an intra-abdominal tumor through a noninvasive process. Larger studies are needed to further validate this method and identify a more precise detection rate. As methods of DNA extraction and sequencing improve, we are hopeful that this may lay the groundwork for an opportunity to detect ovarian cancer at early or even premalignant stages.